L-Homocysteine is a naturally occurring amino acid that functions as a biosynthetic precursor to L-methionine. Applications of homocysteine have been proposed in the treatment of cancer and other serious disorders, increasing its importance as a pharmaceutical or as a pharmaceutical ingredient. Recently, elevated levels of L-homocysteine have also been correlated with the development of coronary artery disease. See Clarke et al, New England Journal of Medicine, 324: 1149-1155 (1991). As a result, many physicians now recommend diets and nutritional supplements to reduce L-homocysteine levels in blood. L-Methionine is an essential amino acid for animals; the racemic form of methionine commands a market of approximately 500 million dollars annually as an additive to animal feed.
The accurate quantitation of L-homocysteine and L-methionine is currently a challenge for analytical laboratories, particularly those analyzing samples of biological fluids. Given the nutritional and medical importance of these amino acids, accurate measurement of their concentrations is often necessary.
Quantitation of both L-homocysteine and L-methionine is especially important in the diagnosis and treatment of homocystinuria. Homocystinuria is a serious genetic metabolic disorder caused most commonly by a block in the pathway of methionine metabolism due to a deficiency of the enzyme cystathionine synthetase. The result is an accumulation of elevated levels of L-homocysteine, L-methionine and metabolites of L-homocysteine in the blood and urine. Homocystinuria is more fully-described in Mudd S H, Levy H L, Skovby F: Disorders of Transsulfuration, in Scriver C R, Beaudet A L, Sly W S, Valle D, eds., The Metabolic and Molecular Basis of Inherited Disease, McGraw-Hill Co., New York, 7th edition, 1995, pp. 1279-1327, the disclosure of which is hereby incorporated by reference.
The ability to detect both L-homocysteine and L-methionine is important in early screening for this disease. Classic homocystinuria, caused by a block in methionine metabolism due to cystathionine synthetase deficiency, is diagnosed by elevated L-methionine and L-homocysteine in blood and urine. In healthy patients, L-methionine and L-homocysteine are generally found in only trace amounts. In patients with classic homocysteinuria, L-methionine levels in the blood are generally above 67 .mu.mol/L, and often above 134 .mu.mol/L, and total L-homocysteine levels in the blood are as high as 500 .mu.mol/L. See Ueland et al., Clinical Chemistry 39: 1764-1779 (1993); and Chace et al., Clinical Chemistry 42: 349-355 (1996), the disclosures of which are incorporated herein by reference. However, variant forms of homocystinuria are caused by lowered N-5-methyltetrahydrofolate homocysteine methyltransferase activity due to vitamin B12 deficiency and decreased N-5,10 methylene tetrahydrofolate reductase activity. These variant forms of homocystinuria are characterized by elevated L-homocysteine and normal or low blood levels L-methionine. Thus, an assay that detects only L-methionine, such as Guthrie microbiological assay that is currently used, can frequently produce false negative results, resulting in mis-diagnosis or delayed diagnosis and treatment.
Such failures or delays have serious, and often fatal, consequences. If untreated, death within the first year of life from homocystinuria is common. Because of the severity of the disease, 21 states currently mandate routine screening of all newborn babies for homocystinuria. The availability of a more reliable test could increase the accuracy in detecting this serious disorder and further broaden use of the test in neonatal screening.
Existing methodology for measuring L-homocysteine and/or L-methionine is poor. Currently, all neonatal screening assays for homocystinuria rely upon the Guthrie microbiological assay. Bacteria which require methionine for growth are cultured from blood specimens taken from the heel of newborn babies. The presence of L-methionine in the sample allows the growth of bacteria on an agar plate, and the diameter of the bacterial growth spot is measured. However, this test is not quantitative, and requires at least 1-2 days for a result. The Guthrie method also produces a high frequency of both false negatives and false positives. See Clinical Chemistry 42: 3, 349-355 (1996), the disclosure of which is hereby incorporated by reference. It is further limited by the fact that it cannot detect L-homocysteine at all. Since homocystinuria is frequently characterized by high levels of L-homocysteine, or its oxidized dimer homocystine, in the absence of high concentrations of L-methionine, the current test is far less reliable than is desired. Furthermore, the existing Guthrie assay does not lend itself to rapid testing for daily management of the levels of L-homocysteine and/or L-methionine, an important aspect of controlling the effects of the disease through diet. A convenient assay with greater reliability would provide significantly improved information to the neonatal physicians, permitting more timely diagnosis and more effective treatment of homocystinuria. Such an assay could also be used by patients and their doctors for daily monitoring of blood or urine levels of L-homocysteine and/or L-methionine, improving daily management of the disease through diet and medications and for other assays of L-homocysteine, L-homocystine (which may be easily reduced to L-homocysteine), and L-methionine.
An assay for L-homocysteine is described in U.S. Pat. No. 5,631,127, which relates to a method for assaying L-homocysteine in a blood, plasma, or urine sample. The method described in this patent comprises the steps of reacting homocysteine with a homocysteine-converting enzyme that also requires at least one substrate other than homocysteine, and without chromatographic separation, assessing a non-labeled analyte selected from a homocysteine co-substrate and the homocysteine conversion products. The example given in this patent is the enzyme S-adenosylhomocysteine hydrolase, and the analyte is adenosine. As adenosine reacts with L-homocysteine to form S-adenosylhomocysteine, the amount of L-homocysteine may be determined by measuring the amount of adenosine consumed, and correlating this with the amount consumed in a set of standard solutions of L-homocysteine. Typically, the change in adenosine concentration is accomplished by immunoassay.
A direct immunoassay for homocysteine is described in U.S. Pat. No. 5,478,729. The method of U.S. Pat. No. 5,478,729 requires chemical modification of the homocysteine to increase its immunogenicity. Antibodies that are specific to the modified homocysteine are prepared, and the modified homocysteine is detected immunologically. This assay is useful in detecting L-homocysteine in the presence of L-cysteine, but the various steps involved in chemical modification and immunological detection, as well as the need for an antibody which is not readily available, make it inconvenient to carry out. Neither of these assays is readily adaptable to doctor's office or home use.
Other assays for detecting L-homocysteine and/or L-methionine are possible, but all have drawbacks. Tandem mass spectrometry on dried blood spots has recently been proposed; this method provides accurate data, but requires very expensive equipment, expensive isotopically-labeled compounds, and highly skilled operators to carry out. Because of its very high cost, this method is probably better suited for confirmation of a preliminary diagnosis made on the basis of a less expensive screening assay. As an alternative, urine or blood samples can be prepared for amino acid chromatography, and L-homocysteine and/or L-methionine is then measured by high performance liquid chromatography (HPLC). Fiskerstrand et al. describe a method of L-homocysteine assay involving fluorescent labeling of serum thiols, followed by HPLC separation and detection of the L-homocysteine derivative from the various other sulfur-containing compounds. See Clin. Chem. 39, 2630271 (1993), the disclosure of which is incorporated herein by reference. However, these methods are time consuming, costly, and not readily available to many laboratories. The cyanide-nitroprusside test may be used to detect homocysteine, but it is non-selective, producing a color change from a non-specific reaction with all sulfhydryl compounds in urine and blood, including cysteine, peptides, proteins, and metabolites. The cyanide-nitroprusside test also gives no reaction at all with L-methionine, and would give false negative results for all homocystinurics producing only symptoms of hypermethionemia. Further, this test is well known to give false positive results on urine for people with cystinuria and acetonuria. See Khashayer Sakhaee and Roger A. L. Sutton, Kidney Stones: Medical and Surgical Management Chapter 46, F. L. Coe et al eds., Lippencott-Raven Publishers, Philadelphia, Pa. (1996), the disclosure of which is hereby incorporated by reference.
Thus, the existing assays for determining the concentration of L-homocysteine and/or L-methionine in biological fluids do not provide the desired selectivity and speed, and frequently give false positive and false negative results. A more quantitative assay that can selectively and rapidly measure the L-homocysteine and/or L-methionine concentration in a biological sample such as urine or blood, without false positive results caused by other sulfhydryl compounds present and without false negative results due to limited sensitivity, would facilitate screening of all newborns for homocystinuria. Further, the assay would be useful in research with homocysteine as an anti-cancer drug, in monitoring L-homocysteine levels in persons at risk for coronary artery disease, and in assessing L-methionine concentrations in pharmaceutical mixtures and nutritional products such as animal feed. Availability of such an assay for these purposes, as well as for the determination of the concentrations of L-homocysteine and/or L-methionine in any other solution, would be greatly desired.